The Effect And Mechanism Of VDR On The Endoplasmic Reticulum Stress Of Acute Kidney Injury

Background:Acute kidney injury(AKI)is a common critically ill syndrome in clinical practice.The ischemia-reperfusion injury(IRI)model is currently the most widely used model in clinical AKI and kidney transplantation research due to its high clinical relevance.IRI is a dynamic process in which the production of reactive oxygen species(ROS)during ischemia and subsequent reoxygenation triggers a series of harmful cellular reactions,leading to inflammation,cell death and acute renal failure.In recent years,with the progress in understanding the potential mechanism of IRI and oxidative stress,more and more studies have proved that endoplasmic reticulum stress(ERS)is of great significance in IRI.IRI can alter the function of the endoplasmic reticulum,leading to the accumulation of unfolded or misfolded proteins,and the resulting ERS can then induce the activation of signal transduction pathways to promote the development of IRI.The endoplasmic reticulum(ER)is the central site for the folding,post translational modification,and transportation of secreted proteins and membrane proteins.Environmental and genetic factors that disrupt the function of the ER lead to the accumulation of misfolded and unfolded proteins in the lumen of the ER,which is known as ERS.Persistent or intense ERS and dysfunction of the unfolded protein response(UPR)pathway ultimately lead to cell death.ERS and disrupted protein deposition lead to the onset of various glomerular and tubular diseases.Vitamin D receptor(VDR)is highly expressed in renal tubular epithelial cells and is a nuclear transcription factor with extensive biological effects.The effect of VDR on ERS in ischemic renal injury has not been reported at home and abroad.Therefore,this topic aims to explore the effect of VDR on ERS in ischemic renal injury and clarify its regulatory mechanism on ERS.Methods:1.The mouse models of acute renal injury induced by ischemia reperfusion(I/R)were established in C57BL/6J mice,Vdr gene knockout mice(Vdr-KO)constructed against the background of C57BL/6J mice,and proximal tubular epithelial cells with Vdr specific overexpression(Vdr-OE)mice.The expression of serum creatinine and blood urea nitrogen in mice was detected,and the pathological changes of renal tissue in mice were detected by HE staining.The morphological changes of endoplasmic reticulum in proximal renal tubular epithelial cells of mice in each group were observed by electron microscopy(EM).Immunohistochemistry or immunofluorescence were used to detect the expression of ERS proteins Bi P,ATF4,ATF6 and CHOP in renal tissue.TUNEL was used to detect apoptosis.Western blot was used to detect the expression levels of VDR,Bi P,CHOP,ATF4,ATF6,and Cleaved-caspase 3 in the renal cortex of mice.2.C57BL/6J mice and Vdr gene knockout mice(Vdr-KO)were used as the background to construct a mouse model of ERS induced acute renal injury induced by tunicamycin(TM).The expression of serum creatinine and blood urea nitrogen in each group of mice was detected,and the pathological changes of renal tissue in mice were detected by HE staining and PAS staining.The morphological changes of endoplasmic reticulum in proximal renal tubular epithelial cells of mice in each group were observed by EM,the expression of ERS protein CHOP in mouse renal tissue was detected by immunofluorescence,apoptosis was detected by TUNEL staining,the expression of IL-1β、 IL-6,MCP-1 and TNF-a in mouse serum were detected by ELISA,and the expression levels of VDR,Bi P,ATF4,ATF6 and CHOP in mouse renal cortex were detected by Western blot.3.The cell models of acute cell injury induced by TM were established in human renal tubular epithelial cells(HK-2).Overexpression of ATF4 by transfection with ATF4 plasmid,silencing the expression of ATF4 or ATF6 by transfecting with ATF4 or ATF6 si RNA.The treatment group was pretreated with VDR agonist Paricalcitol.The morphological changes of cells in each group were observed by phase contrast microscopy,the apoptosis level of cells in each group was detected by TUNEL,and the expression of ATF4,ATF6,CHOP and Cleaved-caspase3 were detected by Western blot.4.Bioinformatics software was used to analyze and predict potential VDR binding sites(VDRE)in the ATF4 and ATF6 promoter regions,and VDRE was verified by chromatin immunoprecipitation(ChIP)and q PCR.Construction of luciferase reporter gene plasmids containing ATF4 or ATF6 promoter sequences,including ATF4 or ATF6 wild-type plasmids(WT-ATF4 or WT-ATF6)and ATF4 or ATF6 mutant plasmids(MUT-ATF4 or MUT-ATF6).The transcriptional regulation mechanism of VDR on ATF4 or ATF6 was determined through dual luciferase reporter gene experiments.Results:1.In the model of acute renal injury induced by ischemia reperfusion(I/R-AKI)established against the background of wild-type C57BL/6J mice,compared with the sham group,the serum creatinine and blood urea nitrogen in the I/R group were significantly increased,the pathological damage of renal tissue was aggravated,cell apoptosis was increased,endoplasmic reticulum damage was aggravated,and the level of ERS was significantly increased.Vdr-KO mice aggravated the above pathological damage and biochemical changes induced by I/R,while pretreatment with VDR agonist Paricalcitol or Vdr-OE could partially reverse these changes.After I/R treatment,the expression of ATF4 and ATF6 in mouse kidney tissue increased,and the expression of ATF4 and ATF6 could be downregulated by Paricalcitol pretreatment.Compared with wild-type mice,the expression of ATF6 in the renal tissue of Vdr-KO mice was upregulated,while the up-regulation of ATF4 was not significant.After I/R treatment,the expression of ATF4 and ATF6 in Vdr-KO mice was significantly higher than that in I/R group,while overexpression of VDR significantly decreased the expression of ATF4 and ATF6 in the renal tissue of mice induced by I/R.2.In the model of tunicamycin induced acute renal injury(TM-AKI)established against the background of wild-type C57BL/6J mice,compared with the control group,the TM group had higher levels of serum creatinine and urea nitrogen,increased renal tissue pathological damage,increased apoptosis,elevated inflammatory indicators,increased endoplasmic reticulum damage,and significantly increased ERS levels,while pretreatment with the VDR agonist Paricalcitol could partially reverse these changes.In the TM-AKI mouse model constructed against the background of Vdr-KO mice,knockout of VDR exacerbated TM induced pathological and biochemical changes.The expression of ATF4 and ATF6 in mouse kidney tissue was significantly increased after TM stimulation,Paricalcitol pretreatment could significantly inhibited the expression of ATF4 and ATF6 induced by TM.Compared with wild type mice,the expression of ATF6 in kidney tissue of Vdr-KO mice was upregulated,while the increase of ATF4 was not significant;after TM stimulation,the expressions of ATF4 and ATF6 in KO+TM group were significantly upregulated compared to TM group.3.After TM stimulation of HK-2 cells,the level of ERS and cell apoptosis significantly increased,while pretreatment with Paricalcitol could partially alleviate these changes;Overexpression of ATF4 weakened the protection of Paricalcitol against TM induced ERS and apoptosis,while silencing the expression of ATF4 or ATF6 enhanced the protective effect of Paricalcitol against TM induced HK-2 cell damage.4.The results predicted by JASPAR software indicate the presence of multiple VDRE in the promoters of ATF4 and ATF6.The results of ChIPq PCR showed that the ATF4 promoter contained three VDRE,and the ATF6 promoter contained one VDRE.The results of the dual luciferase reporter gene experiment showed that the luciferase activity in the group containing the VDR and ATF4 or ATF6 overexpression plasmid was significantly weaker than that in the group containing only wild-type ATF4 or ATF6 reporter gene plasmid,while the luciferase activity intensity did not decreased after mutation of the ATF4 or ATF6 plasmid VDRE.Conclusion:1.VDR plays renal protective role in I/R induced AKI by inhibiting ERS;2.VDR inhibits ERS by downregulating the expression of ATF4 or ATF6 through transcriptional regulation.